This application is a 371 of PCT/EP00/02865 filed Apr. 3, 2000.
The invention relates to compounds having glycoprotein hormone agonistic or antagonistic activity, in particular to compounds having Luteinizing Hormone (LH) agonistic activity. The invention furthermore relates to byciclic heteroaromatic derivative compounds, to pharmaceutical compositions containing the same as well as to the use of these compounds in medical therapy, particularly for use as a control of fertility.
Gonadotropins serve important functions in a variety of bodily functions including metabolism, temperature regulation and the reproductive process. The hypophyseal gonadotropin FSH for example plays a pivotal role in the stimulation of follicle development and maturation whereas LH induces ovulation (Sharp, R. M. Clin Endocrinol. 33:787-807, 1990; Dorrington and Armstrong, Recent Prog. Horm. Res. 35:301-342, 1979). Currently, LH is applied clinically, in combination with FSH, for ovarian stimulation i.e. ovarian hyperstimulation for in vitro fertilisation (IVF) and induction of ovulation in infertile anovulatory women (Insler, V., Int. J. Fertility 33:85-97, 1988, Navot and Rosenwak, J. Vitro Fert. Embryo Transfer 5:3-13, 1988), as well as for male hypogonadism and male infertility.
Gonadotropins act on specific gonadal cell types to initiate ovarian and testicular differentiation and steroidogenesis. The actions of these pituitary and placental hormones are mediated by specific plasma membrane receptors that are members of the large family of G-protein coupled receptors. They consist of a single polypeptide with seven transmembrane domains and are able to interact with the Gs protein, leading to the activation of adenyl cyclase.
Gonadotropins destined for therapeutic purposes can be isolated from human urine sources and are of low purity (Morse et al, Amer. J. Reproduct. Immunol. and Microbiology 17:143, 1988). Alternatively, they can be prepared as recombinant gonadotropins.
As with other therapeutic proteins, it is necessary to administer gonadotropins either subcutaneous or intra-muscular. It would be advantageous, however, to activate the receptor with a small molecule that could be administered through e.g. the oral or transdermal route.
The present invention describes the preparation of such low molecular weight hormone analogs that selectively activate one of the gonadotropin receptors. This should be considered as one of the major advantages of the present invention.
Thus, the invention resides in bicyclic heteroaromatic derivatives according to general formula I, or a pharmaceutically acceptable salt thereof, 
wherein
R1 is NR5R6, OR5, SR5 or R7, preferably R1 is R7;
R5 and R6 are independently selected from H, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (2-7C)heterocycloalkyl, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, (6-14C)aryl or (4-13C)heteroaryl, or R5 and R6 together are joined in a (2-7C)heterocycloalkyl ring;
R7 is (3-8C)cycloalkyl, (2-7C)heterocycloalkyl, (6-14C)aryl or (4-13C)heteroaryl; preferably R7 is (6-14C)aryl or (4-13C)heteroaryl;
R2 is (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, or (6-14C)aryl or (4-13C)heteroaryl, both optionally substituted with one or more substituents selected from (1-8C)alkyl, (1-8C)alkylthio, (1-8C)(di)alkylamino, (1-8C)alkoxy, (2-8C)alkenyl, or (2-8C)alkynyl;
R3 is (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (2-7C)heterocycloalkyl, or (6-14C)aryl or (4-13C)heteroaryl, both optionally substituted with one or more substituents selected from (1-8C)alkyl, (1-8C)(di)alkylamino or (1-8C)alkoxy; preferably R3 is (1-8C)alkyl, more preferably (1-4C)alkyl, even more preferably R3 is isopropyl or tert-butyl;
X is S, O or NR4);
R4 is H, (1-8C)alkyl, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl or (6-14C)aryl(1-8C)alkyl;
Y is CH or N, preferably Y is N;
Z is NH2 or OH;
A is S, N(H), N(R9), O or a bond and
R9 can be selected from the same groups as described for R2 and
B is N(H), O, or a bond.
The alkyl group, alkenyl group or alkynyl group, if present in R5 and/or R6 in the above mentioned formula may optionally be substituted with one or more substituents selected from hydroxyl, (6-14C)aryl, (1-8C)alkoxy, (1-8C)alkylcarbonyloxy, (6-14C)arylcarbonyloxy, (1-8C)alkoxycarbonyl, (6-14C)aryloxycarbonyl, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, amine, (1-8C)alkylaminocarbonyl, (6-14C)alkylaminocarbonyl, (1-8C)alkylcarbonylamino, (6-14C)arylcarbonylamino, (6-14C)(di)arylamino and/or (1-8C)(di)alkylamino.
If R7 is (6-14C)aryl or (4-13C)heteroaryl, aryl may optionally be substituted at the ortho and/or meta position with one or more substituents selected from R8, (6-14C)aryl, (4-13C)heteroaryl, (2-7C)heterocycloalkyl, (3-8C)cycloalkyl, NHR8, OR8 and/or SR8 in which R8 is (6-14C)aryl, (4-13C)heteroaryl, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, the alkyl group of which may be optionally substituted with one or more substituents selected from hydroxyl, (1-8C)alkoxy, (2-7C)heterocycloalkyl((1-8C)alk)oxy, (3-8C)cycloalkyl((1-8C)alk)oxy, (6-14C)aryl((1-8C)alk)oxy, (4-13C)heteroaryl((1-8C)alk)oxy, (2-7C)heterocycloalkyl, (3-8C)cycloalkyl, (6-14C)aryl, (4-13C)heteroaryl, (1-8C)alkoxycarbonyl, (6-14C)aryloxycarbonyl, (1-8C)alkylcarbonyloxy, (6-14C)arylcarbonyloxy, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, amine, (1-8C)alkylaminocarbonyl, (6-14C)alkylaminocarbonyl, (1-8C)alkylcarbonylamino, (6-14C)arylcarbonylamino, (6-14C)(di)arylamino and/or (1-8C)(di)alkylamino. Preferably the substituents at aryl in R7 are chosen from NHR8 or OR8. R8 preferably is (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, (1-8C)alkyl. The most preferred substituents in the alkyl group are (2-7C)heterocycloalkyl, (1-6C)(di)alkylamino and amine.
The alkyl group, alkenyl group or alkynyl group, if present in R9 or R2 in the above mentioned formula may optionally be substituted with one or more substituents selected from (6-14C)aryl, (4-13C)heteroaryl, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, (1-8C)alkylcarbonyloxy, (6-14C)arylcarbonyloxy, (6-14C)aryloxycarbonyl and/or (1-8C)alkoxycarbonyl.
The alkyl group, alkenyl group or alkynyl group, if present in R3 in the above mentioned formula may optionally be substituted with one or more substituents selected from hydroxyl, (1-8C)alkoxy, (6-14C)aryloxy, (3-8C)cycloalkyl((1-8C)alk)oxy, (2-7C)heterocycloalkyl((1-8C)alkoxy, (6-14C)aryl((1-8C)alkoxy, (4-13C)heteroaryl((1-8C)alk)oxy, (2-7C)heterocycloalkyl, (6-14C)aryl, (4-13C)heteroaryl, (1-8C)alkoxycarbonyl, (6-14C)aryloxycarbonyl(1-8C)alkylcarbonyloxy, (6-14C)arylcarbonyloxy, (1-8C)alkylcarbonyl, (6-14C)arylcarbonyl, amine, (1-8C)alkylaminocarbonyl, (6-14C)arylaminocarbonyl, (1-8C)alkylcarbonylamino, (6-14C)arylcarbonylamino, (6-14C)(di)arylamino or (1-8C)(di)alkylamino.
Preferred compounds according to the invention are compounds according to general formula I wherein X is S and/or Z is NH2. Amongst these preferred compounds those wherein X is S and Z is NH2 are especially preferred, even more preferred are those compounds wherein in addition Y is N. Most preferred are the compounds which in addition to the above mentioned definitions of X, Z and Y are defined by R1 being (6-14C)aryl or (4-13C)heteroaryl. Most preferably A is S.
Highly preferred compounds of the invention are the bicyclic heteroaromatic derivative compounds having the general formula I wherein
R1 is (6-14C)aryl or (4-13C)heteroaryl,
R2 is (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, or (6-14C)aryl or (4-13C)heteroaryl, both optionally substituted with one or more substituents selected from (1-8C)alkyl, (1-8C)alkylthio, (1-8C)alkoxy, (2-8C)alkenyl, or (2-8C)alkynyl,
R3 is (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (2-7C)heterocycloalkyl, or (6-14C)aryl or (4-13C)heteroaryl, both optionally substituted with one or more substituents selected from (1-8C)alkyl, (1-8C)(di)alkylamino or (1-8C)alkoxy
X is S, Z is NH2, A is S and B is N(H), O, or a bond.
These compounds have the general structure: 
wherein R1, R2, R3 and B have the above mentioned definitions including the substitutions at the alkyl, alkenyl, alkynyl, aryl or heteroaryl groups in R2, R3. The substitutions of the aryl or heteroaryl groups in R1 are defined previously for R7.
The most preferred compounds are the compounds of general formula I, more preferably formula II, wherein B is N or O, B is N being the most preferred. R2 and/or R3 preferably are (1-8C)alkyl, more preferably (1-4C)alkyl and Y preferably is N.
Particularly preferred compounds according to the invention are those wherein R3 is isopropyl or tert-butyl, tert-butyl being the most preferred.
Excluded from the invention are the compounds ethyl 5-amino-4-phenyl-2-ethoxycarbonylmethylthio-thieno[2,3-d]pyrimidine-6-carboxylate, methyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate, ethyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate, 6-acetyl-5-amino-4-phenyl-2-(2-oxopropylthio)-thieno[2,3-d]pyrimdine, 5-amino-6-benzoyl-4-phenyl-2-phenylcarbonylmethylthio-thieno[2,3-d]pyrimidine or 5-amino-6-(4-chlorobenzoyl)-4-phenyl-2-[(4-chlorophenyl)carbonylmethylthio]-thieno[2,3-d]pyrimidine.
The disclaimer relates to the disclosures in Phosph. Sulf. Sil. Rel. Chem: 60, 223-231, 1991; J. Chem. Res., Synop. (6):290-291, 1998 and Suflir Lett. 9:101-108, 1989.
The term (1-8C)alkyl as used in the definition of formulas I an II means a branched or unbranched alkyl group having 1-8 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, hexyl and octyl. (1-6C)Alkyl groups are preferred, (1-3C)alkyl being the most preferred.
The term (2-8C)alkenyl means a branched or unbranched alkenyl group having 2-8 carbon atoms, such as ethenyl, 2-butenyl etc.
The term (2-8C)alkynyl means a branched or unbranched alkynyl group having 2-8 carbon atoms, such as ethynyl and propynyl.
The term (3-8C)cycloalkyl means a cycloalkyl group having 3-8 carbon atoms, being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclo-octyl.
The term (2-7C)heterocycloalkyl means a heterocycloalkyl group having 3-8 carbon atoms, preferably 3-5 carbon atoms, and at least including one heteroatom selected from N, O or S. Preferred are N or O. Most preferred are piperidine, morpholine and pyrrolidine.
The term (1-8C)alkoxy means an alkoxy group having 1-8 carbon atoms, the alkyl moiety having the same meaning as previously defined. (1-6C)Alkoxy groups are preferred, (1-3C)alkoxy being the most preferred.
The term (1-8C)alkoxycarbonyl means an alkoxycarbonyl group, the alkyl group of which contains 1-8 carbon atoms and has the same meaning as previously defined.
The term (1-8C)(di)alkylamino means an (di)alkylamino group having 1-8 carbon atoms, the alkyl moiety having the same meaning as previously defined.
The term (6-14C)(di)arylamino means an (di)arylamino group having 6-14C carbon atoms, the aryl moiety having the same meaning as previously defined.
The term (1-8C)alkylthio means an alkylthio group having 1-8 carbon atoms, the alkyl moiety having the same meaning as previously defined.
The term (6-14C)aryl means an aromatic hydrocarbon group having 6-14 carbon atoms, such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, anthracyl, which may optionally be substituted with one or more substituents such asxe2x80x94but not limited toxe2x80x94hydroxy, halogen, nitro, trifluoromethyl, cyano, (1-8C)alkylcarbonylamino, (1-8C)alkylamninocarbonyl or (1-8C)(di)alkylamino, the alkyl moieties having the same meaning as previously defined. The preferred aromatic hydrocarbon group is phenyl.
The term (6-14C)aryloxycarbonyl means an aryloxycarbonyl group, the aryl group of which contains 6-14 carbon atoms and has the same meaning as previously defined.
The term (6-14C)aryl(1-8C)alkyl means an arylalkyl group having 7-22 carbon atoms, wherein the alkyl group is a (1-8C)alkyl group and the aryl group is a (6-14C)aryl as previously defined. Phenyl(1-8C)alkyl groups are preferred arylalkyl groups, such as benzyl.
The term (4-13C)heteroaryl means a substituted or unsubstituted aromatic group having 3-13 carbon atoms, preferably 4-9, at least including one heteroatom selected from N, O and/or S, like imidazolyl, thienyl, benzthienyl, quinolyl, tetrahydroquinolyl, isoquinolyl, tetrahydroisoquinolyl, indolyl, acridinolyl, furyl or pyridyl. The substituents on the heteroaryl group may be selected from the group of substituents listed for the aryl group. Preferred heteroaryl groups are thienyl, furyl and pyridyl.
The term joined in a (2-7C)heterocycloalkyl ring in the definition of NR5R6, where R5 and R6 together with the nitrogen atom to which they are bonded are a ring, means a ring containing the nitrogen atom and further having at most 2-7 carbon atoms, which ring may contain unsaturated bonds or one or more heteroatoms selected from N, O and/or S. Examples of such rings are azetidine, pyrrolidine, piperidine, piperazine, morpholine and thiomorpholine.
The term halogen means fluorine, chlorine, bromine or iodine.
The term (2-7C)heterocycloalkyl(1-8C)alkoxy means a heterocycloalkyl group containing 3-8 carbon atoms as defined previously, attached to a (1-8C)alkoxy group, the alkoxy moiety having the meaning as previously defined.
The term (3-8C)cycloalkyl(1-8C)alkoxy means a cycloalkyl group containing 3-8 carbon atoms as defined previously, attached to a (1-8C)alkoxy group, the alkoxy moiety having the meaning as previously defined.
The term (6-14C)aryl(1-8C)alkoxy means an aryl group containing 6-14 carbon atoms as defined previously, attached to a (1-8C)alkoxy group, the alkoxy moiety having the meaning as previously defined. (4-13C)Heteroarylalkoxy groups are analogs of the (6-14C)arylalkoxy groups, at least including one heteroatom selected from N, O and S.
The term (1-8C)alkylcarbonyl means an alkylcarbonyl group, the alkyl group of which contains 1-8 carbon atoms and has the same meaning as previously defined.
The term (6-14C)arylcarbonyl means an arylcarbonyl group, the aryl group of which contains 6-14 carbon atoms and has the same meaning as previously defined.
The term (1-8C)alkylcarbonyloxy means an alkylcarbonyloxy group, the alkyl group of which contains 1-8 carbon atoms and has the same meaning as previously defined.
The term (6-14C)arylcarbonyloxy means an arylcarbonyloxy group, the aryl group of which contains 6-14 carbon atoms and has the same meaning as previously defined.
The term (1-8C)alkylaminocarbonyl means an alkylaminocarbonyl group, the alkyl group of which contains 1-8 carbon atoms and has the same meaning as previously defined.
The term (6-14C)arylaminocarbonyl means an arylaminocarbonyl group, the aryl group of which contains 6-14 carbon atoms and has the same meaning as previously defined.
The term (1-8C)alkylcarbonylamino means an alkylcarbonylamino group, the alkyl group of which contains 1-8 carbon atoms and has the same meaning as previously defined.
The term (6-14C)arylcarbonylamino means an arylcarbonylamino group, the aryl group of which contains 6-14 carbon atoms and has the same meaning as previously defined.
The term (2-7C)heterocycloalkyloxy means a heterocycloalkyl group containing 3-8 carbon atoms as defined previously, attached to an oxygen atom.
The term (3-8C)cycloalkyloxy means a cycloalkyl group containing 3-8 carbon atoms as defined previously, attached to an oxygen atom.
The term (6-14C)aryloxy means an aryl group containing 6-14 carbon atoms as defined previously, attached to an oxygen atom. (4-13C)Heteroaryloxy groups are analogs of the (6-14C)aryloxy groups, at least including one heteroatom selected from N, O and S.
It has been shown that compounds of the above mentioned formula I are capable of binding to the LH recepotor and show agonistic LH activity.
The invention further resides in a pharmaceutical composition comprising a bicyclic heteroaromatic derivative compound or salts thereof having the general formula I.
Pharmaceutical compositions which comprise ethyl 5-amino-4-phenyl-2-ethoxycarbonylmethylthio-thieno[2,3-d]pyrimidine-6-carboxylate, methyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate or ethyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate are within the ambit of the present invention. Thus, the compounds according to the invention can be used in therapy. A further aspect of the invention resides in the use of a bicyclic heteroaromatic compound having the general formula I for the manufacture of a medicament for the control of fertility. Preferably the present compounds are used to activate the LH receptor.
The bicyclic heteroaromatic derivative compounds of this invention may possess one or more chiral carbon atoms. The compounds may therefore be obtained as chirally pure compounds or as a mixture of diastereomers and/or enantiomers. Methods for obtaining the chirally pure compounds are well known in the art, e.g. crystallization or chromatography.
For therapeutic use, salts of the compounds of formula I are those wherein the counterion is pharmaceutically acceptable. However, acid addition salts of bases according to formula I, may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are included within the ambit of the present invention.
Examples of acid addition salts include those derived from mineral acids such as hydrochloric acid, phosphoric acid, sulphuric acid, preferably hydrochloric acid and organic acids like citric acid, tartaric acid, acetic acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, and the like.
Suitable administration routes for the compounds of formula I or pharmaceutically acceptable salts thereof, also referred to herein as the active ingredient are intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal administration. Preferably, the compounds may be administered orally. The exact dose and regimen of administration of the active ingredient, or a pharmaceutical composition thereof, will necessarily be dependent upon the therapeutic effect to be achieved (treatment of infertility; contraception), and may vary with the particular compound, the route of administration, and the age and condition of the individual subject to whom the medicament is to be administered.
In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon adsorption. However, a dosage for humans preferably contains 0.0001-25 mg per kg body weight. The desired dose may be presented as one dose or as multiple subdoses administered at appropriate intervals throughout the day, or, in case of female recipients, as doses to be administered at appropriate daily intervals throughout the menstrual cycle. The dosage as well as the regimen of administration may differ between a female and a male recipient
In case of in vitro or ex vivo applications, like in IVF applications, the compounds of the inventions are to be used in the incubation media in a concentration of approximately 0.01-5 xcexcg/ml.
The present invention thus also relates to pharmaceutical compositions comprising a bicyclic heteroaromatic compound according to formula I, i.e. including pharmaceutical compositions comprising ethyl 5-amino-4-phenyl-2-ethoxycarbonylmethylthio-thieno[2,3-d]pyrimdine-6-carboxylate, methyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate or ethyl 5-amino-4-phenyl-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate, 6-acetyl-5-amino-4-phenyl-2-(2-oxopropylthio)-thieno[2,3-d]pyrimidine, 5-amino-6-benzoyl-4-phenyl-2-phenylcarbonylmethylthio-thieno[2,3-d]pyrimidine or 5-amino-4-chlorobenzoyl)-4-phenyl-2-[(4-chlorophenyl)carbonylmethylthio]-thieno[2,3-d]pyrimidine in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxiliaries must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
Pharmaceutical compositions include those suitable for oral, rectal nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may be prepared by any method well known in the art of pharmacy, for example, using methods such as those described in Gennaro et al., Remington""s Pharmaceutical Sciences (18th ed., Mack Publishing company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture).
Such methods include the step of bringing in association the active ingredient with any auxilliary agent. The auxilliary agent(s), also named accessory ingredients, include those conventional in the art (Gennaro, supra), such as, fillers, binders, diluents, disintegrants, lubricants, colorants, flavoring agents and wetting agents.
Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.
For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example, water prior to use.
Compositions, or formulations, suitable for administration by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.
The bicyclic heteroaromatic derivative compounds of the invention can also be administered in the form of implantable pharmaceutical devices, consisting of a core of active material, encased by a release rate-regulating membrane. Such implants are to be applied subcutaneously or locally, and will release the active ingredient at an approximately constant rate over relatively large periods of time, for instance from weeks to years. Methods for the preparation of implantable pharmaceutical devices as such are known in the art, for example as described in European Patent 0,303,306 (AKZO N.V.).
Thus, the compounds according to the present invention can be used for the same clinical purposes as the native LH, with the advantage that they display altered stability properties and can be administered differently.
The compounds of the present invention wherein B=NH, represented by formula (I-a) can generally be prepared following art-known condensation of an acid of formula (III) with an amine of formula (IV). 
The above reaction is typically conducted at room temperature in a suitable solvent, e.g. an aprotic solvent such as N,N-dimethylformamide or dichloromethane, using a coupling reagent such as O-(benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate (TBTU) or bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and a tertiary base, e.g. N,N-diisopropylethylamine.
Likewise, compounds of formula (I) wherein B=O, being represented by formula (I-b) can be prepared in the same way as described above for compounds of formula (I-a), starting from acids with the general structure (III) and alcohols of formula (V). 
The compounds of formula (I) wherein B is a bond, represented by formula (I-c) can be prepared by condensation of pyridyl chlorides (VI) wherein W=GN or C(O)(OEt) with compounds of general structure (VII) in suitable solvents such as ethanol, methanol or tetrahydrofuran at elevated temperature (50xc2x0 C.) in the presence of a base, e.g. sodium ethoxide, sodium methoxide, potassium carbonate or potassium hydroxide. 
Alternatively, compounds of formula (I-c) wherein X=S, represented by formula (I-d) can also be prepared from thioamides of structure (VIII) wherein W is as previously defined, and compounds of formula (IX) wherein V=halogen such as bromide or chloride, via the abovementioned procedure. 
Related cyclizations are described in literature, see for example Y. A. Sharanin, A. M. Shestopalov and V. K. Promonenkov, J. Org. Chem. USSR (Engl. Transl), 20:1828 1984; Z. H. Klhalil and A. A. Geies, Phosph. Sulf. Silic. Relat. Elem. 60:223, 1991.
A suitable method for the preparation of intermediate acids (III) is the art-known base-mediated saponification of ethyl esters of general structure (X). Saponification takes place in the presence of a base such as lithium hydroxide, potassium hydroxide or sodium hydroxide in a mixture of aqueous dioxane at elevated temperature (80xc2x0 C. to reflux). 
Compounds of formula (X) may be prepared by cyclization of pyridyl chlorides (VI) with HXCH2C(O)OEt as described previously for the synthesis of compounds (I-c). In certain instances, an intermediatexe2x80x94not cyclizedxe2x80x94product can be isolated, which cyclizes upon repeated treatment with base. Alternatively, compounds of formula (X) wherein X=S may also be prepared via the same procedure described for the synthesis of derivatives (I-d), by cyclization of (VIII) with VCH2C(O)OEt (IX) wherein V is as previously defined.
Related cyclizations are found in literature. For example, thieno cyclizations are described by A. A. Santilli, D. H. Kim and S. V. Wanser, J. Heterocycl. Chem. 8:445, 1971; S. Kohra, Y. Tominaga and A. Hosomi, J. Heterocycl. Chem. 25:959, 1988; H. Vieweg, U. Krasselt, N. Bohm, J. Prantz and G. Wagner, Pharmazie 45:731, 1990; H. Vieweg and G. Wagner, Pharmazie 46:51, 1991; G. Wagner, H. Vieweg and S. Leitner, Pharmazie 48:588, 1993. Pyrrolo cyclizations are described e.g. by D. H. Kim and A. A. Santilli, J. Heterocycl. Chem. 6:819, 1969.
Compounds of formula (VI) wherein W is as previously defined, can be synthesized following literature procedures as described for example by A. A. Santilli D. H. Kim and S. V. Wanser, J. Heterocycl. Chem. 8:445, 1971. In a typical experiment, an amide of general structure (XI) is treated with POCl3 at elevated temperature (80xc2x0 C. to reflux). The addition of an appropriate solvent, e.g. dioxane, and/or the addition of either PCl5 or N,N-dimethylaniline to the reaction mixture may result in shorter reaction times and higher yields of chlorides (VI). 
In another approach, amides (XI) may be treated at elevated temperature (preferably reflux) with SOCl2 to give compounds of formula (VI), as was described in literature by D. H. Kim and A. A. Santilli, J. Heterocycl. Chem. 6:819, 1969.
Compounds of formula (VIII) wherein W is as previously defined can be prepared by treatment of derivatives (XI) with a sulfurizing agent, e.g. P2S5 or Lawesson""s Reagent in an appropriate solvent such as pyridine at elevated temperature (preferably reflux), see Z. H. Khalil, Phosph. Sulf. Silic. Relat. Elem. 60: 223, 1991.
Furthermore, compounds of general formula (VIII) wherein Y=CH and A is a bond, represented by formula (VIII-a) can be synthesized by cyclization of xcex1,xcex2-unsaturated ketones of formula (XII) and thioacetamide (XIII). 
In a typical experiment compounds (XII) and (XIII) are reacted in a solvent such as ethanol, methanol or tetrahydrofuran at elevated temperature (preferably reflux) in the presence of base, e.g. piperidine, triethylamine, sodium methoxide or sodium ethoxide. Related cyclizations are found in literature: H. Vieweg, V. Hanfeld, S. Leitner and G. Wagner, Pharmazie 44:639, 1989; H. Vieweg and G. Wagner. Pharmazie 46:51, 1991.
Alternatively, compounds of formula (VIII-a, W=CN) can be synthesized starting from xcex1,xcex2-unsaturated dinitriles of general structure (XIV) and thioacetamides (XV) as was described by G. A. H. Elgemeie, Heterocycles 31:123, 1990. 
Compounds of formula (XI) wherein Y=N, represented by formula (XI-a) can be prepared via several literature-based approaches. 
For example, derivatives of formula (XI-a) wherein R1=(6-14C)aryl or (4-13C)heteroaryl may be synthesized by condensation of ethyl esters (XVI, wherein W is as previously defined, with aldehydes (XVII) and compounds (XVIII), which may be isothiourea (XVIII-a), isourea (XVIII-b), monosubstituted guanidines (XVII-c), disubstituted guanidines (XVIII-d) or amidines (XVIII-e). 
In a typical experiment, components (XVI), (XVII) and (XVIIIa-e) are suspended in an appropriate solvent, e.g. ethanol, methanol, N,N-dimethylformamide, N-methylpyrrolidinone, tetrahydrofuran or pyridine and a base such as potassium carbonate, sodium acetate, sodium methoxide or sodium ethoxide is added. Reaction takes place at elevated temperature (70xc2x0 C. to reflux). After filtration, residues are taken up in water and acidified (pH 2) after which products (XI-a) precipitate (S. Kambe, K. Saito and H. Kishi, Synthesis 287 (1979); A. M. Abd-Elfattah, S. M. Hussain and A. M. El-Reedy, Tetrahedron 39, 3197 (1983); S. M. Hussain, A. A. El-Barbary and S. A. Mansour, J. Heterocycl. Chem. 22, 169 (1985)). In the case of W=C(O)OEt, aromatization occurs on the addition of an oxidant, such as DDQ or oxygen. Related cyclizations may also be performed on a solid support such as Merrifield resin using an appropriate linker, see for example A. L. Mrzinzik and E. R. Felder, J. Org. Chem. 63, 723 (1998); T. Masquelin, D. Sprenger, R. Baer, F. Gerber and Y. Mercadal, Helv. Chim. Acta 81, 646 (1998).
Alternatively, derivatives of formula (XI-a) wherein R1 is not (6-14C)aryl or (4-13C)heteroaryl, may be prepared via substitution of Cl in derivatives of formula (VI-a) or substitution of 4-SMe in compounds of formula (XI-b). 
Related substitution reactions are found in literature, e.g. S. Kohra, Y. Tominaga and A. Hosomi, J. Heterocycl. Chem. 25:959, 1988; A. A. Santilli, D. H. Kim and S. V. Wanser, J. Heterocycl. Chem. 8:445, 1971; J. Clark, M. S. Shannet, D. Korakas and G. Varvounis, J. Heterocycl. Chem. 30:1065, 1993; S. Tumkevicius, Liebigs Ann. Org. Bioorg. Chem. 9:1703, 1995.
Pyridines of general formula (XI) wherein Y=CH, A=S and W=CN, represented by formula (XX) are accessible by sequential alkylation of xcex1,xcex2-unsaturated dinitriles of general structure (XIV) with carbon disulfide and alkyl iodide R2-I to give compounds of general formula (XIX), as described by P. Milart, Tetrahedron 54: 15643-15656, 1998. Cyclization of compounds of formula (XIX) under acidic conditions as described by K. Peseke, Z. Chem. 29:442443 (1989) yields pyridines of general formula (XX). 
Methods to determine receptor binding as well as in vitro and in vivo assays to determine biological activity of gonadotropins are well known. In general, expressed receptor is contacted with the compound to be tested and binding or stimulation or inhibition of a functional response is measured.
To measure a functional response isolated DNA encoding the LH receptor gene, preferably the human receptor, is expressed in suitable host cells. Such a cell might be the Chinese Hamster Ovary cell, but other cells are also suitable. Preferably the cells are of mammalian origin (Jia et al, Mol. Endocrin., 5:759-776, 1991.
Methods to construct recombinant LH expressing cell lines are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition). Expression of receptor is attained by expression of the DNA encoding the desired protein. Techniques for site directed mutagenesis, ligation of additional sequences, PCR, and construction of suitable expression systems are all, by now, well known in the art. Portions or all of the DNA encoding the desired protein can be constructed synthetically using standard solid phase techniques, preferably to include restriction sites for ease of ligation. Suitable control elements for transcription and translation of the included coding sequence can be provided to the DNA coding sequences. As is well known, expression systems are now available which are compatible with a wide variety of hosts, including prokaryotic hosts such as bacteria and eukaryotic hosts such as yeast, plant cells, insect cells, mammalian cells, avian cells and the like.
Cells expressing the receptor are then are then contacted with the test compound to observe binding, or stimulation or inhibition of a functional response.
Alternatively isolated cell membranes containing the expressed receptor may be used to measure binding of compound.
For measurement of binding radioactively or fluorescently labeled compounds may be used. As reference compound human recombinant LH can be used. In the alternative also competition binding assays can be performed.
Another assay involves screening for LH receptor agonist compounds by determining stimulation of receptor mediated cAMP accumulation. Thus, such a method involves expession of the receptor on the cell surface of a host cell and exposing the cell to the test compound. The amount of cAMP is then measured. The level of cAMP will be reduced or increased, depending on the inhibitory or stimulating effect of the test compound upon binding to the receptor.
In addition to direct measurement of e.g. cAMP levels in the exposed cell, cells lines can be used which in addition to transfection with receptor encoding DNA are also trarsfected with a second DNA encoding a reporter gene the expression of which responds to the level of cAMP. Such reporter genes might be cAMP inducible or might be constructed in such a way that they are connected to novel cAMP responsive elements. In general, reporter gene expression might be controlled by any response element reacting to changing levels of cAMP. Suitable reporter genes are e.g. LacZ, alkaline phosphatase, firefly luciferase and green fluorescence protein. The principles of such transactivation assays are well known in the art and are described e.g. in Stratowa, Ch, Himmler, A and Czemilofsky, A. P. (1995) Curr.Opin.Biotechnol. 6:574.
For selecting active compounds testing at 10xe2x88x925 M must result in an activity of more than 20% of the maximal activity when LH is used as a reference. Another criterion might be the EC50 value which must be  less than 10xe2x88x925 M, preferably  less than 10xe2x88x927 M.
The skilled artisan will recognize that desirable EC50 values are dependent on the compound tested. For example, a compound with an EC50 which is less than 10xe2x88x925 M is generally considered a candidate for drug selection. Preferably this value is lower than 10xe2x88x927 M. However, a compound which has a higher EC50, but is selective for the particular receptor, may be even a better candidate.
Screening for LH receptor agonistic compounds can also be performed by using a mouse Leydig cell bioassay (Van Damme, M., Robersen, D. and Diczfalusy, E. (1974). Acta Endocrinol. 77:655-671 Mannaerts, B., Kloosterboer, H. and Schuurs, A. (1987). Neuroendocrinology of reproduction. R. Rolland et al. Eds., Elsevier Science Publishers B.V., 49-58). In this assay, stimulation of LH receptor mediated testosterone production can be measured in Leydig cells isolated from male mice.
To measure in vivo activity of LH receptor agonistic compounds ovulation induction in immature mice can be studied. In this assay immature female mice can be primed with urinary FSH and approximately 48 hours later treated with a LH agonistic compound. The animals are killed after LH agonist treatment and the number of ova in the oviduct can be microscopically assessed.
The compounds of the present invention can be applied clinically in those regimens where now LH or hCG is used. These include LH substitution among subjects with hypogonadal hypogonadism either male or female, midcycle administration to induce ovulation (ovulation induction (OI) or controlled hyperstimulation (COH) or stimulation of the corpus luteum.